Tocols as adjuvant in vaccine

ABSTRACT

Oil based vaccines often cause serious local side effects at the site of administration and/or have a high viscosity which make said vaccines difficult to handle. Vaccines comprising as an adjuvant a stable emulsion of tocols in water do not display these undesired effects and induce good immune responses.

This application is a divisional of application Ser. No. 08/320,202,filed Oct. 7, 1994, which is a continuation of application Ser. No.08/007,400, filed Jan. 21, 1993, now abandoned, which is a continuationof Ser. No. 07/474,434, filed Feb. 2, 1990, now abandoned.

The invention relates to a vaccine which contains a stable emulsion withtocols and to the method for the preparation of such a vaccine.

For protection against transmittable infectious diseases it is customaryto vaccinate humans and animals with immunogenic material against whichprotective antibodies can be formed.

For this purpose, for example, the pathogen itself can be administeredin a live, but preferably non-infectious form, or the killed pathogen oran antigen fraction of the pathogen in which the infectious component islacking can be administered.

In these latter two cases it is necessary also to add to the antigen oneor more components which stimulate the immune response of the organismto be protected. Such immune response-stimulating components are usuallyreferred to by the term adjuvants. Freund's complete adjuvant is awater-in-oil (w/o) emulsion of mineral oil and killed mycobacteria andis regarded as one of the most powerful adjuvants. Other known adjuvantsare emulsions of mineral oils, such as Freund's incomplete adjuvant, andof vegetable oils, such as peanut oil, maize oil, cottonseed oil andsunflower oil, and semi-synthetic oils, such as Miglyol 812N and Myritoloil.

However, the abovementioned emulsions, especially water-in-oil emulsionsbased on mineral oil, cause serious tissue irritations, inflammationswellings and cysts at the site of administration, for which reason theroutine use of these emulsions in humans and animals is less desirableand in certain cases is even prohibited. Moreover, vaccines based on w/oemulsions are relatively viscous, which makes injection of the vaccinemore difficult.

Reducing the oil content causes such a rise in the viscosity that thevaccine can no longer be injected. At the same time this can have aneffect on the stability of the emulsion.

The use of α-tocopheryl acetate as a water-in-oil emulsion in a vaccinefor the protection of rams against infection with Brucella ovis wasdescribed by Afzal et al., Veterinary Immunology and Immunopathology 7(1984), 293-304. However, the vaccine contains approximately 50%dl-α-tocopheryl acetate, as a result of which the vaccine becomesviscous and consequently is difficult to handle.

The aim of the present invention is thus to provide a vaccine which doesnot cause undesired local effects in humans or animals of the typedescribed above and at the same time is easy to handle.

The vaccine according to the invention is characterized in that itcontains a stable emulsion of tocols as adjuvant in water.

Surprisingly it has been found that an oil-in-water emulsion of a tocolderivative of this type couples the favourable characteristic of a lowviscosity, and thus easier handling, with an adjuvant action which is atleast as good as that of a water-in-oil emulsion which contains acorresponding tocol derivative. This result is surprising in view of thefact that it is known that oil-in-water emulsions are less goodadjuvants than water-in-oil emulsions (Herbert, W. J., The mode ofaction of mineral-oil emulsion adjuvants on antibody production in mice,Immunology 14 (1968), 301-318 and Herbert, W. J., Mineral-oil adjuvantsand the immunization of laboratory animals, in: Handbook of experimentalimmunology Vol. 3, ed. by D. M. Weir, third edition, Blackwell 1979).

The vaccine according to the invention consequently contains a stableoil-in-water emulsion with good adjuvant characteristics, causes noadverse local effects after administration and is readily injectable.

Tocols which can be used as adjuvant according to this invention areunderstood to mean tocol and derivatives of tocol. Tocol and derivativeshereof can be represented by the general formula I: ##STR1## wherein Rmay be H or one or more of identical or different substituents chosenfrom the group comprising alkyl, alkoxy, acyloxy, hydroxy, a sulphateand a phosphate group;

R₁ and R₃ independently of one another are H or alkyl;

R₂ is H or alkyl and may be different in each unit;

the broken line indicates the presence or absence of an additionalcarbon-carbon bond in a unit; and

n=has the value 1 to 10.

The alkyl group in R, R₁, R₂ and R₃ may be chosen in particular from alinear or branched carbon chain having 1-4 carbon atoms, such as methyl,ethyl, butyl or isobutyl.

The compound tocol is represented by the formula II: ##STR2##

Tocol derivatives are, inter alia: 5-methyltocol, 7-methyltocol,8-methyltocol, 5,7-dimethyltocol, 5,8-dimethyltocol, 7,8-dimethyltocol,5,7,8-trimethyltocol, 8-methyltocotrienol, 7,8-dimethyltocotrienol,5,8-dimethyltocotrienol, 5,7,8-trimethyltocotrienol, 5,7-diethyltocol,5,7-dimethyl-8-ethyltocol, 5,7-diethyl-8-methyltocol, the esters, suchas formates, acetates, succinates and nicotinates, the sulphates andphosphates, and also the ethers, such as the methyl and ethyl ethers ofthese compounds, and 6-desoxytocol.

A preferred class of tocols to be used in the present invention may berepresented by the general formula III: ##STR3## wherein: R₄ may be H,an acyloxy group, said group preferably being derived from an aliphaticor aromatic carboxylic acid with 1-8 carbon atoms, or a sulphate orphosphate group,

R₅ may be H or one up to three identical or different alkyl groups with1-4 carbon atoms.

More in particular, tocol and esters thereof as well as5,7,8-trimethyltocol (Vitamin-E) and esters thereof according to thegeneral formula III can advantageously be applied in a vaccine accordingto the present invention.

A very suitable tocol derivative according to this invention is,5,7,8-trimethyltocol acetate (α-tocopherol acetate). In practice theracemate, dl-α-tocopherol acetate, is usually employed, although it isalso possible to use the optically active compound.

The concentration of tocols in vaccines according to the invention ispreferably about 0.1-40% by weight and in particular about 2.5-10.0% byweight.

It is also possible to use a mixture of two or more different tocolswith adjuvant action in a vaccine. In addition to tocol or a derivativehereof, the emulsion may contain further components having an adjuvantaction.

Suitable further components are, for example, avridin, carbomers,non-ionic block polymers and muramyl dipeptides.

An emulsifier which can be used in the stable emulsion according to thisinvention can be chosen from the group of emulsifiers which arecustomarily used for the present purpose. Suitable emulsifiers are,inter alia, non-ionic surfactants, such as polyoxyethylene sorbitanmono-oleate, polyoxyethylene monolaurate, polyoxyethylene fatty acidesters, such as polyoxyethylene stearate, polyoxyalkyl ethers, such aspolyoxyethylene cetyl ether, polyoxyethylene castor oil derivatives,polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose,lethicin and gelatin; anionic surfactants including salts of alkylsulphate esters, such as sodium lauryl sulphate; cationic surfactantsand amphoteric surfactants. The concentration of the emulsifieraccording to the invention is preferably between 0.1 and 20% and moreparticularly between 2.5 and 7.5% by weight.

The vaccine may be prepared by means of emulsifying tocols withimmunogenic material containing an aqueous solvent. In anotherembodiment the vaccine is prepared by means of emulsifying tocols withan aqueous solvent, after which the emulsion is mixed with immunogenicmaterial. It is also possible, for the preparation of a vaccineaccording to the invention, to use as the starting material tocols towhich immunogenic material has been added, which are then emulsifiedwith an aqueous solvent.

The vaccine is preferably prepared by mixing tocols with an emulsifier,after which this mixture is emulsified with water. Immunogenic materialis added to the emulsion thus obtained, by which means the desiredstable emulsion is finally formed. It is desirable for the physicalstability of the vaccine that the dispersed particles of tocols have acertain size. It has been found that the physical stability manifestsitself best if the dispersed particles are preferably smaller than 20 μmand more particularly smaller than 1 μm.

The vaccine according to the invention is suitable for use of live andnon-live material as immunogen. Non-live antigen material may beselected from killed pathogens or immunogenic fractions (subunits)thereof. The vaccine is also suitable for generating an immune responseagainst non-pathogenic substances, for example for the production ofantisera for use in diagnostic tests and for immunological sterilizationor castration.

The pathogens can be, for example: viruses, bacteria or parasites. Thesecan be killed by chemical or by physical means. In this context killingsignifies inactivation, for example by such a change in the geneticmaterial and/or other vital components that the pathogen is no longercapable of reproduction. Suitable chemical agents for killing pathogensare, for example, formaldehyde, glutaraldehyde, β-propiolactone,ethyleneimine and derivatives, or another compound which can reactbifunctionally or multifunctionally with reactive groups belonging tothe pathogen. Physical agents for killing pathogens are, for example, UVradiation, γ radiation, "heat shock" and X-radiation.

Antigen fractions of the abovementioned pathogens or of non-pathogenicsubstances can be obtained therefrom by means of chemical or physicalmethods, and, if desired, followed by separation of a fraction with theaid of chromatography, centrifuging and similar techniques. After theseoperations components are obtained which, although very pure if desired,frequently will also have poor immunogenicity. If desired, thesecomponents of pathogens or non-pathogenic substances can be bound to acarrier (for example "keyhole limpet" hemocyanin or liposomes or othermicellar complexes) in order to increase the immunogenicity. Theabovementioned term "immunogenic fractions of pathogens ornon-pathogenic substances" is also used to signify: synthetic antigensor haptens, with which natural antigens of the pathogen ornon-pathogenic substances in question are imitated. Synthetic antigensor haptens of this type can be prepared in a known manner with the aidof organic synthetic methods or in the case of, for example,polypeptides with the aid of recombinant DNA methods.

The concentration of antigen in a vaccine according to the invention isin general 1-85% by weight.

In addition to a vaccine of this type which contains immunogenicmaterial from only one pathogen (so-called monovalent vaccines),vaccines which contain immunogenic material from several pathogens(so-called combination vaccines) likewise fall within the scope of theinvention. Combination vaccines of this type contain, for example,material from various viruses or from various strains of a single virusor from virus/bacterium combinations or various bacteria. Combinationvaccines of this type can also contain material from parasites.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows virus neutralizing tilers at specified time intervals aftervaccination.

FIG. 2 depicts virus liters in nasal swabs at daily intervals aftervirus challenge.

FIG. 3 shows body weights at daily time intervals before and after viruschallenge.

EXAMPLE 1

To prepare a vaccine against Gumboro's disease, 7.5 g of vitamin Eacetate and 5.0 g of Polysorbate 80 are mixed together with continuousstirring. This mixture is then made up to 50 g with distilled water. Thevaccine is prepared by mixing 50 g of the above mixture with 50 g of anaqueous solution of inactivated immunogenic material, with stirring, sothat the vaccine finally contains 18.2% (weight per unit volume) Gumborovirus antigen.

EXAMPLE 2

Three groups of 10 four-week-old SPF chickens were vaccinatedintramuscularly with 0.5 ml of vaccine containing inactivated Gumborovirus (10⁷,3 TCID₅₀ /ml) prepared according to Example 1. Blood wastaken 4, 8, 12, 16 and 20 weeks after vaccination, after which antibodytitres were determined in the serum by means of an ELISA (incubationwith virus-coated microtitre plate+chicken serum; incubation withanti-chicken-Ig's antibodies-enzyme conjugate).

    __________________________________________________________________________            Antibody response after (weeks post-vaccination)                      Vaccine 4     8     12    16    20                                            __________________________________________________________________________    saline solution                                                                         7.2 ± 2.2.sup.a                                                                 7.1 ± 2.2                                                                       6.8 ± 1.6                                                                        7.3 ± 1.7                                                                        7.6 ± 1.4                                  vit. E acetate.sup.b                                                                  12.6 ± 2.1                                                                       11.5 ± 1.3                                                                       9.3 ± 1.5                                                                        9.4 ± 1.3                                                                        9.0 ± 1.3                                  mineral oil.sup.c                                                                     10.0 ± 1.3                                                                       11.9 ± 1.4                                                                       11.5 ± 0.9                                                                       11.4 ± 0.7                                                                       10.9 ± 1.1                                 __________________________________________________________________________     .sup.a) mean ELISA titre (.sup.2 log) with standard deviation                 .sup.b) 7.5% by weight o/w emulsion                                           .sup.c) Freund's incomplete w/o emulsion.                                

EXAMPLE 3

Two groups of 10 five-week-old mice were vaccinated intramuscularly with0.1 ml of vaccine containing inactivated Aujeszky virus, preparedanalogously to Example 1 (10⁸ TCID₅₀ /ml). Blood was taken 4, 8 and 12weeks after vaccination, after which antibody titres were determined inthe serum by means of an ELISA (incubation with virus-coated microtitreplate+mouse serum; incubation with anti-mouse-Ig's antibodies-enzymeconjugate).

    ______________________________________                                                 Antibody response after                                                       (weeks post-vaccination)                                             Vaccine    4            8        12                                           ______________________________________                                        saline solution                                                                          11.7 ± 1.2.sup.a                                                                        11.2 ± 1.3                                                                          11.3 ± 1.6                                vit. E acetate.sup.b                                                                     13.2 ± 1.2                                                                              13.1 ± 1.8                                                                          13.1 ± 1.5                                ______________________________________                                         .sup.a mean ELISA titre (.sup.2 log) with standard deviation                  .sup.b 7.5% by weight o/w emulsion.                                      

EXAMPLE 4

Groups of 10 four-week-old SPF chickens were vaccinated intramuscularlywith 0.5 ml of vaccine containing purified E. coli-F11-pilus protein (20μg/dose), prepared analogously to Example 1. Blood was taken 4, 8, 12and 16 weeks after vaccination, after which antibody titres weredetermined in the serum by means of an ELISA (incubation with E.coli-F11-pilus-protein-coated microtitre plate+chicken serum; incubationwith anti-chicken-Ig's antibodies-enzyme conjugate).

    ______________________________________                                                Antibody response after                                                       (weeks post-vaccination)                                              Vaccine   4         8         12     16                                       ______________________________________                                        saline solution                                                                         5.9 ± 1.4.sup.a                                                                       8.3 ± 2.4                                                                            8.6 ± 2.2                                                                         7.2 ± 0.8                            vit. E-acetate.sup.b                                                                    10.5 ± 0.8                                                                           14.5 ± 0.8                                                                           13.3 ± 0.9                                                                        12.0 ± 1.6                            mineral oil.sup.c                                                                       9.9 ± 2.8                                                                            13.5 ± 2.3                                                                           13.5 ± 1.8                                                                        12.3 ± 1.3                            ______________________________________                                         .sup.a mean ELISA titre (.sup.2 log) with standard deviation                  .sup.b 7.5% by weight o/w emulsion                                            .sup.c Freund's incomplete w/o emulsion.                                 

EXAMPLE 5

In the same experiment as described in Example 4 chickens were alsovaccinated with a water-in-oil emulsion with vitamin E acetate, based oncastor oil. The result of this is compared with the oil-in-wateremulsion with vitamin E acetate from Example 4.

    ______________________________________                                        Antibody response after (weeks post-vaccination)                              Vaccine                                                                              4         8        12     16     20                                    ______________________________________                                        w/o-     9.8 ± 1.1.sup.a                                                                    11.8 ± 1.5                                                                          12.0 ± 1.6                                                                        10.6 ± 1.2                                                                         9.0 ± 1.1                         emulsion.sup.b                                                                o/w-   10.5 ± 0.8                                                                           14.5 ± 0.8                                                                          13.3 ± 0.9                                                                        12.0 ± 1.6                                                                        10.0 ± 1.1                         emulsion.sup.c                                                                ______________________________________                                         .sup.a) mean ELISA titre (.sup.2 log) with standard deviation                 .sup.b) 7.5% vitamin E acetate + castor oil (50%)                             .sup.c) 7.5% vitamin E acetate.                                          

EXAMPLE 6

This example is to demonstrate the adjuvant activity of tocol andderivates thereof in a stable oil-in-water emulsion. The adjuvantpotency of said compounds is illustrated in the table shown below usingthe purified E. coli-FII-pilus protein as an antigen. The experimentswere carried out exactly as described in Example 4.

    ______________________________________                                                  Antibody response after                                                       (weeks post-vaccination)                                            Vaccine     4          8          12                                          ______________________________________                                        saline solution                                                                           7,7 ± 1,6.sup.a                                                                       9,0 ± 1,9                                                                             9,0 ± 1,6                                vit. E acetate.sup.b                                                                      12,1 ± 0,7                                                                            12,4 ± 0,7                                                                            11,8 ± 0,9                               vit. E nicotinate.sup.b                                                                   10,2 ± 1,5                                                                            11,9 ± 1,1                                                                            11,6 ± 1,0                               tocol.sup.b 12,0 ± 1,7                                                                            11,9 ± 0,6                                                                            11,4 ± 0,8                               controls    5,4 ± 0,6                                                                             5,9 ± 1,1                                                                             6,6 ± 1,0                                ______________________________________                                    

EXAMPLE 7

Groups of 5 pigs seronegative for pseudorabies and 4-6 weeks of age werehoused in an isolation unit. Pigs were vaccinated once with a livepseudorabies vaccine (PRV strain 783) having a titre of the livecomponent of 10⁶ TCID₅₀ /dose. In all vaccinations one dose of 2 ml wasgiven intramuscularly behind the ear.

As solvent for the live freeze-dried vaccines:

aqueous diluent (Diluvac, commercially available from Intervet:dil)

mineral oil o/w emulsion (commercially available from Duphar:o/w)

Vitamin-E acetate o/w emulsion (7,5% by weight Vit. E acetate):GFA, wereused.

Blood samples were taken at the intervals indicated in FIG. 1. Serumsamples were prepared and tested for the presence of virus neutralizingantibodies by ELISA. The challenge with 7 log TCID₅₀ of virulentpseudorabies strain 75V19 was done intranasally at 16 weeks aftervaccination. Virus excretion and weight gain were monitored.

RESULTS Virus neutralizing antibodies

FIG. 1 shows the presence of virus neutralizing (VN) antibodies as aresult of a vaccination with the three vaccines mentioned above. At thetime of challenge the VN titre induced by the Vitamin-E acetate o/wvaccine was higher than those induced by the mineral oil o/w vaccine orthe vaccine containing the aqueous diluent.

Virus excretion

Nasal swabs were taken daily after challenge until at least on twoconsecutive days all the animals of a given group were shown to benegative in the test. The viral titres presented as TCID₅₀ per ml ofnasal washing are shown in FIG. 2. Live PRV vaccine with a Vitamin-Eacetate o/w adjuvant shows reduced virus titre, indicating anotherpositive effect of this kind of adjuvant for live vaccines.

Weight gain

Body weights were determined at regular intervals before challenge, andfrom day of challenge body weights were measured daily (FIG. 3).Calculations were performed to obtain the avarage daily weight gain (orloss) in percentage over the 7 days following challenge in accordancewith the method prescribed by the European Pharmacopoeia (Draft EuropeanPharmacopoeia, Nov. 1988, Freeze-dried Aujeszky's disease live vaccinefor pigs) as presented in the table below:

    ______________________________________                                                                    Δ average percentage of                                                 growth per day (between                           Ranking Vaccine    Remarks  day 0 and 7 p.c.)                                 ______________________________________                                        1       783        GFA       1.55                                             2       783        o/w       1.39                                             3       783        Diluvac   0.78                                             4       Controls   --        0.00   (-1.45)                                   ______________________________________                                    

The differences of each group to the control group is given. The animalsof the control groups suffered a weight loss of 1.45% per day during 7days (shown between brackets).

From FIG. 3 and the table it should be concluded that the Vitamine-E o/wadjuvant is more effective in the live vaccine than the mineral oil o/wvaccine which in turn is more effective than the aqueous diluent.

We claim:
 1. A method for immunizing an animal against a particularpathogen, comprising administering to said animal a vaccine comprisingan immunogenic amount of antigen of said particular pathogen in anessentially mineral oil-free, stable oil-in-water emulsion comprisingtocols, wherein the vaccine contains from 0.1 to 40% by weight tocolsand the tocols have the formula: ##STR4## wherein: R may be H or one ormore of identical or different substituents chosen from the groupconsisting of alkyl, alkoxy, acyloxy, hydroxy, a sulphate and aphosphate group;R₁ and R₃ independently of one another are H or alkyl;R₂ is H or alkyl and may be different in each unit; the broken lineindicates the presence or absence of an additional carbon-carbon bond ina unit; and n has the value 1 to
 10. 2. The method of claim 1, whereinthe tocols used in said vaccine are compounds with the formula: ##STR5##wherein: R₄ may be hydroxy, an acyloxy group, a sulfate or a phosphategroup; andR₅ may be H or from one to three identical or different alkylgroups with 1-4 carbon atoms.
 3. The method according to claim 1,wherein the tocol is 5,7,8-trimethyltocol acetate.
 4. The methodaccording to claim 1, wherein the vaccine further comprises at least oneother component having adjuvant action.
 5. The method according to claim1, wherein the vaccine contains from 2.5 to 10% by weight tocols.
 6. Themethod according to claim 1, wherein the tocols are in the form ofdispersed particles and said particles are no larger than 20 μm.
 7. Themethod according to claim 6, wherein said particles are no larger than 1μm.
 8. The method of claim 1, wherein said vaccine is administeredintramuscularly.
 9. The method of claim 1, wherein said vaccine is acombination vaccine.